The vocal folds of the human larynx are highly specialized structures that are capable of self-sustained oscillation for production of sound for speech, communication, and singing. The vocal folds are divided anatomically into three tissue layers (Hirano M. Otologia (Fukuoka) 1975; 21:239-442). The superficial layer is the vocal fold epithelium, followed by the middle lamina propria layer, and the deep muscular layer (FIG. 1). The epithelial layer is very thin compared to the other layers and acts biomechanically as a functional unit with the lamina propria layer and thus these two layers are combined and called the “cover” layer in biomechanical studies of the vocal fold. The lamina propria layer is an amorphous, paucicellular layer composed mostly of fibroblasts, macrophages and extracellular matrix molecules (Gray et al. Laryngoscope 1999; 109:845-54). This layer provides the appropriate viscoelasticity (mucosal pliability) for normal oscillation of the vocal folds during phonation which can be appreciated clinically as mucosal waves on the vocal fold surface upon videostroboscopic examination of the larynx (Hirano et al. J Voice 2009; 23(4):399-407). Vocal fold scarring is a pathologic condition that results from loss of the lamina propria layer and leads to glottic insufficiency and diminished or absent mucosal waves on the vocal fold surface, and is a common clinical problem resulting in dysphonia. Treatment of lamina propria loss is of special interest because there is currently no effective replacement therapy.
The most frequent etiology for vocal fold lamina propria loss is surgery on the vocal fold for benign and malignant disorders. Other causes include a variety of traumatic, neoplastic, iatrogenic, inflammatory, and idiopathic disorders (Rosen Otolaryngol Clin North Am 2000; 33:1081-6). The resulting voice is often rough and breathy, of poor quality, and perceived by patients as a severe communication handicap. Histologically, lamina propria loss after iatrogenic vocal fold injury occurs as the layer is replaced with dense and disorganized collagen deposits (Hirano Curr Opin Otolaryngol Head Neck Surg. 2005 June; 13(3):143-7). This increased fibrosis is referred to clinically as vocal fold scar.
Vocal fold scar is a challenging problem for the otolaryngologist because effective therapy for this condition is currently lacking and rehabilitation of patients is difficult. Management of vocal fold scars with autologous fat implantation and autologous fascia augmentation has been reported but treatment results have been less than satisfactory (Benninger et al. Otolaryngol Head Neck Surg 1996; 115:474-82; Neuenschwander et al. J Voice 2001; 15:295-304; Duke et al. Laryngoscope 2001; 111:759-64). This is because currently no biomaterial exists that matches the native viscoelastic properties of this layer.
There are currently two potential therapeutic modalities for vocal fold scars: (a) Injection of biomaterials into the lamina propria compartment, and (b) cellular based therapy (Hsiung et al. Laryngoscope 2000; 110:1026-33; Chhetri et al. Otolaryngol Head Neck Surg 2004; 131: 864-70). The biomaterial approach has been problematic because durable biomaterials that also have the appropriate viscoelastic properties have yet to be developed. Current biomaterials are all too stiff to be injected into the lamina propria compartment.
The ability to take cells from an individual, expand those cells in the laboratory, and inject them into the same individual to repair symptomatic tissue defects is a newly evolving therapeutic modality in medicine. Initial work in this area of “cultured autologous cellular” therapy was directed towards chondrocytes. A Federal Drug Administration (FDA) approved autologous cultured chondrocyte product (Carticel®, Genzymebiosurgery, Cambridge, Mass.) has been available since 1997 for orthopedic use (Hayflick L and Moorhead P S. Exp Cell Research 1961; 25:585-621). Autologous cellular therapy has also been applied clinically to include transplantation of autologous cultured melanocytes for treatment of segmental vitiligo (Treco D A et al. Fibroblast cell biology and gene therapy. In: Chang P L, ed. Somatic Gene Therapy. Boca Raton: CRC Press, 1995:49-60), autologous keratinocytes for treatment of ulcers and burns (Jacobson et al. Am J Speech Lang Pathol 1997; 6:66-70; Hartnick et al. Laryngoscope 2005; 115:4-15), and autologous fibroblasts for treatment of facial wrinkles (Boss et al. Ann Plast Surg 2000; 44:536-42; Watson et al. Arch Facial Plast Surg 1999; 1:165-70; Kanemaru et al Ann Otol Rhinol Laryngol 2003; 112:915-920). No adverse effects such as malignant transformation of injected cells or significant tissue reaction have been reported so far with the use of autologous cellular therapy.
The lamina propria layer is composed primarily of ECM molecules such as collagen, elastin, and proteoglycans. Fibroblast cells in this layer produce these ECM molecules (Gray, et al., 1999). Theoretically, injection of autologous fibroblasts into the lamina propria layer could lead to reconstitution of normal lamina propria ECM components and improve the voice disorder by re-establishing normal mucosal pliability of the vocal folds. Lamina propria replacement therapy must consider the complex three dimensional organization of this layer that develops and matures over a significant time period. At birth, the lamina propria layer is a hyper-cellular monolayer and it matures over the next seven to thirteen years into the trilaminar layer with the differential fiber and proteoglycan composition seen in adults. This three dimensional organization is complex with not only differences in the relative composition of the ECM molecules within the layer but also in the three dimensional orientation of collagen and elastic fibers. Lamina propria replacement therapy therefore needs to address not only the viscoelasticity of the replacement material but its three-dimensional geometry and composition as well. Production of such lamina propria with its normal components in proper concentration and configuration remains a daunting task with currently available tissue engineering techniques.
Fibroblasts are readily obtained from skin or buccal mucosa by punch biopsy and can be cultured free of other cell types. Human fibroblasts do not spontaneously become immortal in culture, a property that has significant implications when their re-injection into a human being is considered. Cultured autologous fibroblast therapy in humans has so far been directed mainly in the cosmetic plastic surgery field for the treatment of facial wrinkles and scars (Boss et al. Ann Plast Surg 2000; 44:536-42; Watson et al. Arch Facial Plast Surg 1999; 1:165-70; Kanemaru et al. Ann Otol Rhinol Laryngol 2003; 112:915-920). Boss and colleagues reported treating 1,000 subjects with cultured autologous fibroblasts. They performed approximately 4,000 injections from 1995 through 1999. The follow-up period was 36-48 months. Ninety-two percent of the subjects were satisfied with the therapy. There were a total of 13 reported reactions (0.27%) to the injections, of which 11 were mild reactions with redness and swelling that resolved within 48-72 hours. The other two reactions were moderate with swelling and erythema for 7-10 days. Watson and colleagues reported a 6-month prospective pilot study in 10 adults to assess the efficacy of cultured autologous fibroblasts to treat skin wrinkles and dermal depressions. Microscopic examination of the injection site was also performed and demonstrated a denser and thicker layer of collagen in the dermal region, absence of any inflammatory reaction, and viable fibroblasts throughout. No adverse reactions were noted clinically or microscopically.
A canine study has previously shown the potential for autologous fibroblast injection therapy for treatment of vocal fold scars. Canine lamina propria replacement therapy was performed where autologous fibroblasts were harvested from buccal mucosal biopsies and expanded in the laboratory. Fourth or fifth passage fibroblasts were injected into previously scarified vocal folds. The scarred vocal folds had absent or severely limited mucosal waves and poor acoustic parameters. Significant improvements in mucosal waves and acoustic parameters were obtained following autologous fibroblasts injection therapy. Chhetri D K et al. Otolaryngol Head Neck Surg 2004; 131: 864-70). Two months later less atrophy was observed in the treated vocal fold compared to the control vocal fold. Histologically the injected cells appeared viable. However, phonation studies were not performed and the mucosal wave grade or the acoustic quality of voice was not provided. In another study, human embryonic stem cells were injected into scarred rabbit vocal folds and histologic assessment performed one month later (Cedervall, et. al. Laryngoscope 2007; 117:2075-2081). Persistence of the embryonic stem cells was observed and the injected vocal fold was associated with decreased viscoelasticity (as measured by parallel plate rheometry) as compared to the untreated but scarred side. Another study performed injection of autologous fibroblasts from skin into scarred rabbit vocal folds. In contrast from other reports, this study “primed” the fibroblasts by addition of various factors such as epidermal growth factor, hepatocyte growth factor (HGF), and decorin to the cell culture (Krishna et al. Otolaryngol Head Neck Surg 2006; 135:937-945). They reported that HGF treated cells demonstrated increased synthesis of hyaluronic acid, and the HGF and decorin treated cells demonstrated diminished collagen synthesis in vivo.
While demonstration of appropriate changes to the ECM components is important, ultimately the resulting vibratory behavior of the vocal folds is most important. Specifically, the return of vocal fold mucosal pliability as improved mucosal waves upon phonation should be one of the ultimate criteria for success. One previous animal study has performed autologous fibroblast injection into scarred vocal folds in a canine model and demonstrated return of mucosal waves and improved acoustic parameters (Cedervall et al. Laryngoscope 2007; 117:2075-2081). In that study, vocal fold scarring was induced unilaterally in the animals and resulted in absent or severely limited mucosal waves and significantly worse acoustic parameters. Autologous fibroblasts were harvested from the canine buccal mucosa and the cell population was expanded in the laboratory. Fourth or fifth passage fibroblasts were then injected into previously scarified vocal folds. Significant improvements in mucosal waves and acoustic parameters were obtained after lamina propria replacement therapy. After therapy, mucosal waves became normal in four animals and near normal in the other four. No statistical difference was found in mucosal wave grade between baseline and post-therapy. All animals tolerated therapy without complications. Histological analysis of the treated vocal folds demonstrated an increased density of fibroblasts, collagen, and reticulin, a decreased density of elastin, and no change in hyaluronic acid.
It is therefore an object of the present invention to provide defined dosage unit formulations of autologous dermal fibroblasts for injection into human patients for the repair and long term augmentation of vocal cord defects.
It is a further object of the present invention to provide dosage unit formulation that contain stem cells, precursor cells or partially differentiated cells that can be used for the repair and long term augmentation of vocal cord defects in humans.